The present invention is related to the detection of bacteria, such as Mycobacteria, in human or animal body fluids such as blood, sputum and urine. The present invention provides a method for assessing the viability of bacteria such as Mycobacterium tuberculosis without the need for propagation of the bacteria.
For example, tuberculosis (TB) caused by Mycobacterium tuberculosis is a major public health problem in many countries world-wide with particular significance in developing countries. Tuberculosis control programmes are faced with an increased burden of cases, a shift towards diagnostically more difficult categories of patients such as extrapulmonary and smear-negative cases, and the emergence of multidrug-resistant strains of M. tuberculosis. Improved diagnosis would be a valuable contribution in the struggle to solve this global public health emergency.
The method of the present invention is concerned with the amplification of specific nucleic acid sequences.
Nucleic acid amplification reactions promise to reduce the time for diagnosis from weeks to hours, while surpassing the sensitivity and specificity of the classical methods. Besides their potential value in diagnosis, amplification reactions offer the possibility of a rapid identification and drug-susceptibility determination. Amplification of DNA target molecules to a detectable level by the polymerase chain reaction (PCR) is the best analyzed system for detecting Mycobacteria.
The xe2x80x9cPolymerase Chain Reactionxe2x80x9d (PCR) is described in European patent applications EP 200362 and EP 201148. PCR is a cyclic process which has double stranded DNA as target. Each cycle in the PCR process starts with the separation of a double stranded DNA target in its two complementary strands. To each strand a primer will anneal and DNA polymerases present will extend the primers along the DNA strand to which it annealed thus forming two new DNA duplexes. When the reaction mixture is heated the strands of the DNA duplexes will be separated again and a new PCR cycle can start. Thus, the PCR process produces multiple DNA copies of a DNA target. Amplification using PCR, can also be based on an RNA template. The actual PCR needs to be preceded by a reverse transcription step to copy the RNA into DNA (RT-PCR). However, if RT-PCR is used for the detection of transcripts differentiation of mRNA- and DNA-derived PCR products is necessary. DNAse treatment prior to RT-PCR can be employed (Bitsch, A. et al., J Infect. Dis 167, 740-743., 1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271., 1994), but sometimes fails to remove contaminating DNA sufficiently [Bitsch, A. et al., 1993].
More recently a different class of nucleic acid amplification methods namely the xe2x80x9ctranscription based amplification techniquesxe2x80x9d was developed. The techniques involve the transcription of multiple RNA copies from a template comprising a promoter recognized by an RNA polymerase. Said copies are used as input for further amplification. Such methods have been described by Gingeras et al. in WO88/10315 and Burg et al. in WO89/1050. Isothermal transcription based amplification techniques have been described by Davey et al. in EP 323822 (relating to the NASBA method), by Gingeras et al. in EP 373960 and by Kacian et al. in EP 408295 (the TMA method). Transcription based amplification reactions may also be performed with thermostable enzymes. Such a thermostable method is described in EP 682121 filed in the name of Toyo Boseki KK.
The isothermal transcription based nucleic acid amplification techniques have been utilized to detect mycobacteria, such as the NASBA method [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van, Schepers, P. and Klatser, P. R. (1993) Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J. Gen. Microbiol. 139, 2423-2429.] and another transcription-mediated RNA amplification test (TMA)[Jonas, V., Alden, M. J., Curry, J. I., Kamisango, K., Knott, C. A., Lankford, R., Wolfe, J. and Moore, D. F. (1993) Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31, 241] both targeted at 16S ribosomal RNA.
Amplification reactions targeted at the 16S rRNA or the gene encoding it are usually directed to a conserved region which comprises species-specific variable sequences [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van, Schepers, P. and Klatser, P. R. (1993) Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J. Gen. Microbiol. 139, 2423-2429., Jonas, V., Alden, M. J., Curry, J. I., Kamisango, K., Knott, C. A., Lankford, R., Wolfe, J. and Moore, D. F. (1993) Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rRNA. J. Clin. Microbiol. 31, 241]. They have the advantage that a single amplification reaction can identify the mycobacterial species. An additional advantage of the transcription-mediated RNA amplification assays targeted at 16S rRNA, is the high number of target molecules per cellxe2x88x92xc2x12000; sensitivity is thereby favoured.
Since RNA, especially mRNA, has a generally much shorter half-life time than DNA, its detection may be useful for the assessment of the viability of mycobacteria [Moore, D. F., Curry, J. I., Knott, C. A. and Jonas, V. (1996) Amplification of rRNA for assessment of treatment response of pulmonary tuberculosis patients during antimicrobial therapy. J. Clin. Microbiol. 34, 1745-1749., Vliet, G. M. E. van der, Schepers, P., Schukkink, R. A. F., Gemen, B. van and Klatser, P. R. (1994) Assessment of mycobacterial viability by RNA amplification. Antimicrob. Agents Chemother. 38, 1959-1965.], which is relevant to the problems of resistance against drugs and contagiousness of the patient.
The present invention is based on the detection of mRNA encoding the elongation factor EF-Tu.
The elongation factor EF-Tu is essential in (myco)bacterial translation. Elongation factors play an ancillary role in the elongation step of translation and are thus an indicator of the cell""s metabolic activity. For every translation EF-Tu is required. The amount of EF-Tu protein can be as high as 50% of their total protein content in active proliferating cells.
EF-Tu encoding gene sequences (DNA) have been used as a marker to detect the presence of bacterial cells.
In EP133288 a method is disclosed for the detection of bacterial DNA with a probe comprising a base sequence of at least a portion of one of the strands of a tuf or fus gene. Southern blot hybridization of the digested mycoplasmal DNAs with the elongation factor (EF-Tu) gene tuf of E.coli was used as a basis to detect polymorphism in mycoplasma strains.[Yogev et al. FEMS Microbiol.Lett., 50(2-3), 145-9, 1988].
A PCR based assays for the detection of Mycoplasma tuberculosis, and Mycoplasma fermentans using the gene encoding elongation factor Tu (tuf) as the target sequence had also been described [Berg et al.,Mol.Cell.Probes, 10(1), 7-14, 1996 and Luneberg et al., J.Clin.Microbiol., 31(5), 1088-94, 1993].
The present invention, however, is concerned with the detection of EF-Tu mRNA as a marker for bacterial viability.
The present invention thus provides a method for the assessment of bacterial viability whereby mRNA coding for the elongation factor EF-Tu is used as a target in a nucleic acid amplification reaction and the presence and/or amount of said mRNA is determined.
The presumably short-lived mRNA coding for the EF-Tu is most likely highly abundant in the (myco)bacterial cell and a decrease therein will indicate a decline in metabolic activity. Furthermore, because of the EF-Tu""s essential role, it is plausible to assume that it is present in all mycobacterial species, allowing the development of a general amplification system with species-specific primers and/or probes, analogous to the 16S rRNA NASBA design [Vliet, G. M. E. van der, Schukkink, R. A. F., Gemen, B. van, Schepers, P. and Klatser, P. R. (1993) Nucleic acid sequence-based amplification (NASBA) for the identification of mycobacteria. J. Gen. Microbiol. 139, 2423-24291.].
With the method of the present invention, preferably a transcription based amplification technique, such a NASBA, is used for the amplification of the bacterial EF-Tu mRNA. In contrast to RT-PCR, NASBA, which is based on RNA transcription by T7 RNA polymerase (Kievits et al., 1991; Compton, 1991), does not need differentiation between RNA- and DNA-derived amplification products since it uses RNA as its principal target.
The amplified products may be detected using a complementary labeled probe.
Numerous protocols have been described for the detection of amplified products [Klatser, P R. (1995) Amplification reactions in mycobacteriology. J. Microbiol. Meth. 23, 75-87.]. Preferably homogeneous assays are used, because they would allow amplification reaction mixtures to be sealed before amplification is initiated. One such system, electro-chemiluminescence (ECL), has already been succesfully applied to detect amplified products in NASBA [Gemen, B. van, Beuningen, R. van, Nabbe, A., Strijp, D. van, Jurriaans, S., Lens, P., Kievits, T. (1994) A one-tube quantitative HIV-I RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J. Virol. Methods, 49, 157-167.].
The method of the present invention is in particular useful for assessing the viability of Mycobacteria species such as are M. tuberculosis or M. leprae. 
With the present invention oligonucleotides are also provided that can be used as primers and probes for the amplification of bacterial EF-Tu mRNA.
The use of the oligonucleotides according to the invention is not limited to any particular amplification technique or any particular modification thereof. It is evident that the oligonucleotides according to the invention find their use in many different nucleic acid amplification techniques and various methods for detecting the presence of (amplified) bacterial EF-Tu mRNA. The oligonucleotides of the present invention can likewise be used in quantitative amplification methods. An example of such quantitative method is described in EP 525882.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides. Such oligonucleotides may be used as primers and probes.
Of course, based on the sequences of the oligonucleotides of the present invention, analogues of oligonucleotides can also be prepared. Such analogues may constitute alternative structures such as xe2x80x9cPNAxe2x80x9d (molecules with a peptide-like backbone instead of the phosphate sugar backbone of normal nucleic acid) or the like. It is evident that these alternative structures, representing the sequences of the present invention are likewise part of the present invention.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced synthetically, which is capable of acting as a point of initiation of synthesis of a primer extension product which is complementary to a nucleic acid strand (template or target sequence) when placed under suitable conditions (e.g. buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization, such as DNA dependent or RNA dependent polymerase. A primer must be sufficiently long to prime the synthesis of extension products in the presence of an agent for polymerization. A typical primer contains at least about 10 nucleotides in length of a sequence substantially complementary or homologous to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-26 nucleotides but longer primers may also be employed, especially when the primers contain additional sequences such as a promoter sequence for a particular polymerase.
Normally a set of primers will consist of at least two primers, one xe2x80x98upstreamxe2x80x99 and one xe2x80x98downstreamxe2x80x99 primer which together define the amplificate (the sequence that will be amplified using said primers).
Primarily for the use in transcription based amplification techniques, the oligonucleotides according to the invention may also be linked to a promoter sequence. The term xe2x80x9cpromoter sequencexe2x80x9d defines a region of a nucleic acid sequence that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription by which an RNA transcript is produced. In principle any promoter sequence may be employed for which there is a known and available polymerase that is capable of recognizing the initiation sequence. Known and useful promoters are those that are recognized by certain bacteriophage RNA polymerases such as bacteriophage T3, T7 or SP6. Oligonucleotides linked to a promoter sequence are commonly referred to as xe2x80x9cpromoter primersxe2x80x9d.
Oligonucleotides according to the invention are substantially complementary to a sequence of a bacterial EF-Tu mRNA sequence, said oligonucleotide being 10-50 nucleotides in length and comprising at least 10 consecutive nucleotides of one of the sequences depicted in SEQ ID 1-8 or the complementary sequence thereof.
Oligonucleotides comprising (a part of) SEQ ID""s 1 and 2 have proven to be suitable for the amplification of Ef-Tu mRNA sequences of M. tuberculosis. In the event amplification is carried out with a transcription based amplification technique one of said oligonucleotides may also comprise a promoter sequence. Of course, such xe2x80x9cpromoter-oligonucleotidesxe2x80x9d are likewise art of the invention. In the sequence as depicted in SEQ ID 9 the T7 promoter has been linked to the sequence as depicted in SEQ ID 1. In the experimental part of this application these sequences are indicated as TUF15 (SEQ ID 9) and TUF 18 (SEQ ID 2).
The present invention also provides a pair of oligonucleotides for the amplification of M. leprae derived EF-Tu sequences. This pair consists of an oligonucleotide comprising at least 10 consecutive nucleotides of the sequence as. depicted in SEQ ID 3 and an oligonucleotide comprising at least 10 consecutive nucleotides of the sequence as depicted in SEQ ID 4 respectively.
Again, for use in transcription based methods, one of the oligonucleotides may be linked to a promoter sequence, and an oligonucleotide provided with the T7 promoter sequence is depicted in SEQ ID 10. In the experimental part of the application such a pair is referred to as TUF 20 (SEQ ID 10) and TUF 22 (SEQ ID 4).
The present invention further provides a pair of oligonucleotides that are suitable for the amplification of E. coli derived EF-Tu sequences.
This pair consists of an oligonucleotide comprising at least 10 consecutive nucleotides of the sequence. as depicted in SEQ ID 5 and an oligonucleotide comprising at least 10 consecutive nucleotides of the sequence as depicted in SEQ ID 6 respectively.
Again, for use in transcription based methods, one of the oligonucleotides may be linked to a promoter sequence, and an oligonucleotide provided with. the T7 promoter sequence is depicted in SEQ ID 11. In the experimental part of the application such a pair is referred to as TUF 27 (SEQ ID. 11) and TUF 27 (SEQ ID 6).
These oligonucleotides are thus especially useful in the assessment of the viability of M.tuberculosis, M.Leprae or E.coli. 
It is understood that oligonucleotides consisting of the sequences of the present invention may contain minor deletions, additions and/or substitutions of nucleic acid bases, to the extent that such alterations do not negatively affect the yield or product obtained to a significant degree. Where oligonucleotides according to the present invention are used as probes, the alterations should not result in lowering the hybridization efficiency of the probe.
For example, in case of transcription based amplification techniques, wherein one or more of the primers may be provided with a promoter sequence, the introduction of a purine-rich (=G or A) hybridizing sequence, just after the promoter sequence may have positive effects on the transcription (when there are C""s and T""s abortive transcription may occur). If no such sequence is available in the target nucleic acid a purine-rich sequence can be inserted in the oligonucleotide just following the last three G residues of the promoter sequence.
The sequences of the present invention are reflected as DNA sequences. The RNA equivalents of these sequences are likewise part of the present invention.
Part of the oligonucleotides according to the invention are particularly suitable for use as a probe in the detection of nucleic acid amplified with a pair of oligonucleotides according to the invention. When used as a probe, said oligonucleotides may be provided with a detectable label. Various labeling moieties are known in the art. Said moiety may, for example, either be a radioactive compound, a detectable enzyme (e.g. horse radish peroxidase (HRP)), a hapten like biotin, or any other moiety capable of generating a detectable signal such as a calorimetric, fluorescent, chemiluminescent or electrochemiluminescent signal.
Hybrids between oligonucleotides according to the invention and (amplified) target nucleic acid may also be detected by other methods known to those skilled in the art. Oligonucleotides according to the invention that are especially suitable as a probe for the detection of Mycobacterial Ef-Tu sequences consist essentially of the sequences as depicted in SEQ ID 7 and 8 (In the experimental part said sequences are depicted as TUF 25 and TUF 26 respectively.).
Together these probes can be used in a sandwich hybridization assay, whereby one can be used as capture probe and the other can be labeled with a detectable label.
A test kit for the detection of Mycobacterial EF-Tu mRNA in a sample is likewise part of the present invention. Such a kit may comprise
a pair of oligonucleotides according to the invention and at least one oligonucleotide comprising a nucleic acid sequence substantially complementary to at least part of the amplified nucleic acid sequence, provided with a detectable label, as well as suitable amplification reagents.
These reagents are for example the suitable enzymes for carrying out the amplification reaction. A kit, adapted for use with NASBA for example, may contain suitable amounts of reverse transcriptase, RNase H and T7 RNA polymerase. Said enzymes may be present in the kit in a buffered solution but can likewise be provided as a lyophilized composition, for example, a lyophilized spherical particle. Such lyophilized particles have been disclosed in PCT appl. no. WO95/27721. The kit may further be furnished with buffer compositions, suitable for carrying out an amplification reaction. Said buffers may be optimized for the particular amplification technique for which the kit is intended as well as for use with the particular oligonucleotides that are provided with the kit. In transcription based amplification techniques, such as NASBA, said buffers may contain, for example, DMSO, which enhances the amplification reaction (as is disclosed in PCT appl. no. WO 91/02818). 
Furthermore the kit may be provided with an internal control as a check on the amplification procedure and to prevent the occurrence of false negative test results due to failures in the amplification procedure. The use of internal controls in transcription based amplification techniques is described in PCT appl. no. WO 94/04706. An optimal control sequence is selected in such a way that it will not compete with the target nucleic acid in the amplification reaction. Kits may also contain reagents for the isolation of nucleic acid from biological specimen prior to amplification. A suitable method for the isolation of nucleic acid is disclosed in EP389063.